Rapid sample collection in supercritical fluid chromatography

ABSTRACT

A process and device for chromatography, such as preparative and analytical supercritical fluid chromatography, or supercritical fluid extraction for a liquid phase SFC collection system that separates and retains eluted components from sample injections. The process efficiently separates liquid and gas phases in the flow stream upstream of a collection vessel and without additional pressure schemes or solvent extractions imposed on the flowstream. Sample fractions are collected from sample component concentration peaks based upon the chromatographic elution of the sample components. Samples are repeatedly the injected into the mobile phase flow stream and collected into large volume collection containers, thereby allowing longer unattended run times and cost-efficient sample purification and recovery.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation in part of application Ser. No. 09/607,316, APPARATUS AND METHOD FOR PREPARATIVE SUPERCRITICAL FLUID CHROMATOGRAPHY, filed Jun. 26, 2000.

BACKGROUND OF THE INVENTION

[0002] A substantial need exists for industries to recover purified components of interest from samples containing simple or complex mixtures of components. Many technologies have been developed to meet this need. For dissolvable, nonvolatile components, the technology of choice has been liquid elution chromatography.

[0003] Analysts have several objectives in employing preparative elution chromatography. First, they wish to achieve the highest available purity of each component of interest. Second, they wish to recover the maximum amount of the components of interest. Third, they wish to process sequential, possibly unrelated samples as quickly as possible and without contamination from prior samples. Finally, it is frequently desirable to recover samples in a form that is rapidly convertible either to the pure, solvent-free component or to a solution of known composition which may or may not include the original collection solvent.

[0004] In the case of normal phase chromatography, where only organic solvents or mixtures are used as eluants, typical fraction volumes of tens to hundreds of millimeters are common. The fraction must then be evaporated over substantial time to recover the component residues of interest. In reversed phase chromatography, where mixtures of organic solvents and water are used as the elution mobile phase, a secondary problem arises. After removal of lower boiling solvents, recovered fractions must undergo a water removal step lasting from overnight to several days. Thus, availability of the recovered components of interest is delayed by hours or days, even after the separation process is complete. This latter problem can create a serious bottleneck in the entire purification process when enough samples are queued.

[0005] Where difficult separation conditions exist or separation speed is a requirement, a subset of elution chromatography, known as high performance liquid chromatography (HPLC), is preferred. This HPLC technique is used both as an analytical means to identify individual components and as a preparative means of purifying and collecting these components.

[0006] For analytical HPLC, samples with component levels in the nanogram to microgram range are typical. Preparative HPLC systems typically deal with microgram to multiple gram quantities of components per separation. Preparative HPLC systems also require a means to collect and store individual fractions. This is commonly performed, either manually or automatically, simply by diverting the system flow stream to a series of open containers.

[0007] Drawbacks exist to the current use of preparative HPLC. Elution periods ranging from several minutes to hours are necessary for each sample. Further, even in optimal conditions only a small fraction of the mobile phase contains components of interest. This can lead to very large volumes of waste mobile phase being generated in normal operation of the system.

[0008] An alternative separation technology called supercritical fluid chromatography (SFC) has advanced over the past decade. SFC uses highly compressible mobile phases, which typically employ carbon dioxide (CO2) as a principle component. In addition to CO2, the mobile phase frequently contains an organic solvent modifier, which adjusts the polarity of the mobile phase for optimum chromatographic performance. Since different components of a sample may require different levels of organic modifier to elute rapidly, a common technique is to continuously vary the mobile phase composition by linearly increasing the organic modifier content. This technique is called gradient elution.

[0009] SFC has been proven to have superior speed and resolving power compared to traditional HPLC for analytical applications. This results from the dramatically improved diffusion rates of solutes in SFC mobile phases compared to HPLC mobile phases. Separations have been accomplished as much as an order of magnitude faster using SFC instruments compared to HPLC instruments using the same chromatographic column. A key factor to optimizing SFC separations is the ability to independently control flow, density and composition of the mobile phase over the course of the separation.

[0010] SFC instruments used with gradient elution also reequillibrate much more rapidly than corresponding HPLC systems. As a result, they are ready for processing the next sample after a shorter period of time. A common gradient range for gradient SFC methods might occur in the range of 2% to 60% composition of the organic modifier.

[0011] It is worth noting that SFC instruments, while designed to operate in regions of temperature and pressure above the critical point of CO2, are typically not restricted from operation well below the critical point. In this lower region, especially when organic modifiers are used, chromatographic behavior remains superior to traditional HPLC and often cannot be distinguished from true supercritical operation.

[0012] In analytical SFC, once the separation has been performed and detected, the highly compressed mobile phase is directed through a decompression step to a flow stream. During decompression, the CO2 component of the mobile phase is allowed to expand dramatically and revert to the gas phase. The expansion and subsequent phase change of the CO2 tends to have a dramatic cooling effect on the waste stream components. If care is not taken, solid CO2, known as dry ice, may result and clog the waste stream. To prevent this occurrence, heat is typically added to the flow stream. At the low flow rates of typically analytical systems only a minor amount of heat is required.

[0013] While the CO2 component of the SFC mobile phase converts readily to a gaseous state, moderately heated liquid organic modifiers typically remain in a liquid phase. In general, dissolved samples carried through SFC system also remain dissolved in the liquid organic modifier phase.

[0014] The principle that simple decompression of the mobile phase in SFC separates the stream into two fractions has great importance with regard to use the technique in a preparative manner. Removal of the gaseous CO2 phase, which constitutes 50% to 95% of the mobile phase during normal operation, greatly reduces the liquid collection volume for each component and thereby reduces the post-chromatograhic processing necessary for recovery of separated components.

[0015] A second analytical purification technique similar to SFC is supercritical fluid extraction (SFE). Generally, in this technique, the goal is to separate one or more components of interest from a solid matrix. SFE is a bulk separation technique, which does not necessarily attempt to separate individually the components, extracted from the solid matrix. Typically, a secondary chromatographic step is required to determine individual components. Nevertheless, SFE shares the common goal with prep of SFC of collecting and recovering dissolved components of interest from supercritical flow stream. As a result, a collection device suitable for preparative SFC should also be suitable for SFE techniques.

[0016] Expanding the technique of analytical SFC to allow preparative SFC requires several adaptations to the instrument. First the system requires increased flow capacity. Flows ranging from 20 ml/min to 200 ml/min are suitable for separation of multi-milligram up to gram quantities of materials. Also, a larger separation column is required. Finally, a collection system must be developed that will allow, at a minimum, collection of a single fraction of the flow stream which contains a substantially purified component of interest. In addition, there frequently exists a compelling economic incentive to allow multiple fraction collections from a single extracted sample. The modified system must also be able to be rapidly reinitialized either manually or automatically to allow subsequent sample injection followed by fraction collection.

[0017] Several commercial instances of preparative SPC instrumentation have been attempted which have employed different levels of technology to solve the problems of collection. A representative sampling of these products includes offerings from Gilson, Thar, Novasep, and ProChrome. However, no current implementation succeeds in providing high recovery, high purity, and low carryover from sample to sample. For example, one system may use the unsophisticated method of simply spraying the collection stream directly into a large bottle, which results in high sample loss, presumably due to aerosol formation. Another system uses a cyclonic separator to separate the two streams, but provides no rapid or automated means of washing the separators to prevent carryover. Such instruments are typically employed to separate large quantities of material by repetitive injection so that no sample-to-sample cleaning step is required. Other systems use a collection solvent to trap a sample fraction into a volume of special solvent in a collection container. This technique uses relatively large quantities of hazardous solvents to perform sample collection, is prone to sample fraction concentration losses or degradation, and possible matrix interferences exist between fractionated samples and collection solvent constituents.

[0018] An example of a SFC system is illustrated outside of the outlined section 10 in FIG. 1. The schematic flow diagram is a packed-column supercritical fluid chromatography (SFC) system from initial modifier supply to a detector. The system has a carbon dioxide supply tank 200, line chiller 220, pump 202, modifier tank 204 and pump 206, dampener and pressure transducer 208, leading to a mixing column 210, connected to an injection valve 212 that is connected to at least one packed chromatography column 214, and a detector 216.

[0019] In a SFC system, liquefied compressed carbon dioxide gas is supplied from cylinders 200. High pressure tubing 218 connects the carbon dioxide reservoir tank 200 to the carbon dioxide pump 202. The tubing may be cooled 220 prior to connecting to the pump 202. The system uses two HPLC-type reciprocating pumps 202, 206. One pump 202 delivers carbon dioxide and the other pump 206 delivers modifier 204, such as methanol. The carbon dioxide and modifier are combined, creating a mixture of modifier dissolved into the supercritical fluid.

[0020] The combined supercritical fluid is pumped at a controlled mass-flow rate from the mixing column 210 through transfer tubing at a fixed-loop injector 212 where the sample of interest is injected into the flow system. The sample combines with the compressed modifier fluid inside the injection valve 212 and discharges into at least one packed chromatography column 214. After fractionation of the sample occurs in the columns 214, the elution mixture passes from the column outlet into a detector 216.

[0021] SFC is finding significant advantages in the separation of enantiomers and is supplanting normal-phase HPLC for performing chiral separations. The majority of chiral separation by SFC have been performed using packed columns with binary or ternary mobile phases. SFC can also be used as an alternative to the reversed-phase HPLC or polar organic modes used with columns in HPLC. Extremely rapid separations, up to less than a minute, with relatively high resolution are possible. Using binary mobile phases, SFC has been used to derivatize highly polar compounds in order to elute less polar entities with binary mobile phase. Tertiary mobile phases are often used in chiral separations since most are performed in the pharmaceutical industry, and a large fraction of drugs, drug intermediates and metabolites are aliphatic or polyfunctional amines.

SUMMARY OF THE INVENTION

[0022] The present invention relates to sample recovery after separation by supercritical fluid chromatography or supercritical fluid extraction, and improvements therein. More specifically, the present invention provides a method and device to collects fractionated components of sample solutes into one or more collection containers in a more efficient and faster manner while generating less spent solvents for disposal.

[0023] More specifically, the present invention relates to optimally separating a liquid phase, containing sample components of interest, from a much larger gaseous phase after the controlled expansion, or decompression, of a single chromatographic mobile phase from a high working pressure to a lower pressure where it is unstable. The controlled decompression causes a phase separation between liquid and gaseous phases while at the same time aerosol formation is strongly suppressed within the transfer tubing.

[0024] It is a further object of the present invention to provide a device and method to separate monophasic fluids that are mixtures of highly compressed or liquefied gasses and organic liquid modifiers into gaseous and liquid phases inside transfer tubing prior to collection of fractions of the liquid phase into one or more unique collection chambers. The collection of fractions of the liquid phase into collection chambers minimizes liquid solvent use and waste through efficient gas and liquid phase separation prior to entering collection chambers. The collection technique uses no additional solvents for collection of fractions.

[0025] This invention provides a cassette bank of multiple chambers to collect and store separated or extracted fractions. Each collection cassette includes one or more collection chambers, and each chamber can receive a purified liquid fraction. Each chamber may hold a removable sample collection liner. The collection liners may be individually removed, substituted, stored, cleaned and re-used, or discarded. One purpose of the collection liner is to provide a simplified means of transporting the collected liquid fraction from the cassette. A second purpose of the collection liner is to provide a means to eliminate cross-contamination of consecutive samples by providing an easily replaceable, uncontaminated liner in each collection chamber for each sample.

[0026] The present invention manually or automatically controls one or more valves and a sealing mechanism for collection chambers such that multiple liquid phase fractions from one sample may be collected into one or more chambers without mechanically adjusting the collection chamber seals. This method allows for rapid switching between collection chambers in the event of closely separated peaks in the chromatographic flow stream.

[0027] It is a further object of the present invention to facilitate a manual or automatic reset of the collection system to allow consecutive samples to be processed in a rapid manner. Technical difficulties arise in the implementation of a collection system that satisfies all the analysts objectives stated above. The major problem centers around the tremendous expansion (typically 500-fold) of the pressurized liquid or supercritical CO2 fraction of the mobile phase that violently transforms into a gas at atmospheric pressure. This transition has four major negative effects with regard to liquid phase sample collection.

[0028] First, as mentioned above, the expanding CO2 causes a severe temperature drop that has the possibility of forming dry ice and clogging the system. Since flows of preparative SFC systems are much higher than corresponding analytical systems, considerable more heat must be added to compensate for the temperature drop. Care must be taken; however, not to allow the actual temperature to rise in the flow system since this may cause damage to thermally unstable compounds of interest. Higher organic modifier content reduces the severity of this problem, both by adding heat capacity and by dissolving the CO2, thereby preventing dry ice formation.

[0029] Second, as the CO2 expands, it rapidly loses any solvating power it had in the compressed state. If components of interest are largely dependent on the CO2 for solubility they will lose their primary means of transport through the flow system. Solid components will accumulate and eventually clog the flow path causing system failure. Again, the organic modifier component is an important factor here since the liquid will continue to solvate the components of interest and transport them to a collection device. Care must be taken not to introduce too much heat into the flow stream as to drive the organic modified also into the gas phase, otherwise its beneficial effect of transporting the solutes will be lost.

[0030] Third, it is beneficial to complete the transition from liquid to gaseous CO2 in as short a period as possible after the initial decompression stage. While in the liquid state, CO2 can disperse the organic modifier containing components of interest even when it is not dense enough to have any significant solvating power. The dispersion can have the effect of remixing components that had been efficiently separated by the SFC process prior to decompression. The faster the CO2 can be converted the less chromatographic degradation can occur. Two factors seem to predominate in controlling the ability to volatilize the liquid phase CO2: (a) efficient heat transfer between the heat source and the flowing liquid, and (b) residence time of the CO2 in the heated region. The first factor can be positively affected by selection of a highly conductive material such as copper for heater fabrication. Insuring excellent thermal contact between the heater and a thin-walled transfer tubing also facilitates heat transfer to the flowing fluids. Residence time of the decompressing fluid can be controlled by stepping the pressure drop over a series of one or more restrictors in the transfer line. Higher backpressure slows the linear velocity of the biphasic fluid in the heater. So long as the back pressure generated by these restrictions do not interfere with the SFC density regulation in the high pressure separation region, a great deal of turnability is possible for optimizing heat transfer.

[0031] Fourth, due to the expansion, linear velocities of the depressurizing fluid increase dramatically in the transfer tubing. Residual liquids of the system are moved along the flow path largely by shear forces from the expanding gas. This turbulent environment is ideal for the creation of aerosols, whereby very small droplets of modifier liquid are entrained in the gas phase as a “mist.” It is a finding of this study that the aerosol formation within the transfer tubing can be almost completely controlled by proper temperature control of the expanding two-phase system. Aerosol formation is a greater problem at lower temperatures. It is a surprising finding of this work that higher levels of organic modifier with correspondingly lower CO2 content require higher temperature levels to prevent visible aerosol formation.

[0032] In the preferred exemplary embodiment, the SFC collection system is composed of a moderately restrictive, thermally regulated stainless steel transfer tube which extends from a back pressure regulation component of the SFC chromatograph into a multi-port distribution valve and from the valve to a variety of flow paths leading either through discrete collection chambers or directly connected to a vented common waste container.

[0033] Initial separation of the liquid phase sample from carbon dioxide gas occurs immediately at the point of initial decompression within the backpressure regulator of the SFC or SFE instrument. By providing downstream restriction, a minimum backpressure sufficient to prevent the formation of solid CO2 can be maintained while liquid CO2 is present in the transfer lines.

[0034] The remainder of the CO2 evaporation and separation from the organic modifier occurs in the stainless steel transfer tubing prior to entering the cassette. This is accomplished by exposing the transfer tubing to a series of one or more heaters designed to optimize thermal transfer to the fluid. Ideally, this heater series transfers sufficient energy to the liquid CO2 portion of the emerging fluid to allow for complete evaporation of the liquid CO2 and raise the fluid temperature sufficiently to prevent the transfer tubing from icing externally. Because rates of heat transfer are time dependent, it is beneficial to slow the velocity of fluids within the heater series.

[0035] During the CO2 evaporation process within the first heated zone, significant separation between the gaseous CO2 and liquid modifier occurs. However, the separation to pure CO2 and pure organic modifier is never realized for several reasons. First, some organic modifier is typically also evaporated into the gas state. The degree of evaporation is largely dependent on the absolute temperature of the fluids within the transfer tubing. While organic modifier evaporation does lead to lower recovery of liquid phase, it does not necessarily reduce the recovery of dissolved components of interest which do not typically have low enough boiling points to convert to vapor. Second, a fraction of CO2 will remain dissolved in the organic liquid. Both temperature and pressure determine the amount of residual CO2. Higher temperatures reduce CO2 solubility while higher pressures increase CO2 solubility.

[0036] Aerosol formation of the liquid phase is a common problem in SFC sample collection and is a primary cause of loss of the organic liquid phase that contains the dissolved components of interest. Higher temperatures reduce the aerosol generation. The composition of the separated phases also is a factor. Higher temperatures are required to eliminate aerosols in streams with higher organic liquid composition. An additional heated zone is used to trim the fluid temperature to control aerosols. In addition, this heater provides a fine level of temperature control of the fluid before collection in the pressurized collection chamber. As mentioned above, a secondary effect is that a higher trim temperature can reduce the concentration of dissolved CO2, thereby reducing the possibility of uncontrolled or explosive outgassing of the CO2 when the pressure is removed from the collection chamber.

[0037] Following the trim heater, a valve system is used to divert the biphasic flow stream sequentially to waste or to one of the collection chambers in a collection cassette. The valve system is comprised of one or more valves and an electronic controller. The system is designed to offer rapid response to a manual or automated start/stop signal. Typically, the signal would result from detection of a component of interest emerging from the high-pressure flow system. A start signal would be generated at the initial detection of the component while a stop signal would be generated at the loss of detection. The effect of a start signal is to divert the flow to the first unused collection chamber of the cassette. The effect of the stop signal is to divert the flow to waste. Another possible type of start/stop signal may be based on a timetable rather than physical detection of components. The controller may also have features to limit the access time or flow volume allowed to an individual chamber. In addition, the controller may allow or prevent the system from cycling back to the original chamber if more fractions are desired than there exists available collection chambers.

[0038] The collection cassette is a resealable apparatus that contains one or more hollow collection chambers open at the top. In the preferred exemplary embodiment, each chamber holds a removable inert liner. The liner collects a fraction of the original sample dissolved in a liquid solvent base. A preferred exemplary embodiment of a cassette has four chambers housing four test tube vials that function as chamber liners. The number of chambers in a cassette may be varied with no effect on performance. Each test tube vial may hold up to its capacity of a separated sample fraction from the high-pressure flow stream.

[0039] In the preferred embodiment, sample fractions are collected in one chamber of the cassette at a time. The biphasic fluid enters a chamber via a transfer line from the valve system. The tip of the transfer line is preferentially positioned tangential to the inner wall of the collection tube and with a slight downward angle, usually less than 45 degrees from horizontal. Attached to the transfer line and suspended inside a test tube is a guiding spring wire. The spring wire is bowed away from the transfer line and functions as a guide for the transfer line as it descends into a vial. When transfer tubing is properly inserted into a test tube vial, the bowed section of the spring wire engages the circumferential edge of the open end of a test tube vial. As the tubing continues into the test tube, the spring wire compresses against the inner surface of the test tube vial and pushes the tubing toward the opposite side of the vial. As a result, the angled tip of the transfer tubing is pressed against the inner wall of the test tube vial.

[0040] Both the organic liquid and CO2 gas follow a descending spiral path along the inner wall to the bottom of the collection liner. The CO2 gas continues in a path up the center of the liner to a vent in the collection chamber. A restrictive transfer line attached to the vent causes the CO2 gas to pressurize the collection chamber both inside and outside the collection liner. The degree of black pressurization within the chamber is roughly proportional to the composition of CO2 in the original mobile phase.

[0041] The pressurization of the collection chamber serves to slow down the velocity of the CO2 entering the chamber. This in turn reduces the magnitude of shear forces occurring between the CO2 gas and the collected liquid at the bottom of the liner. With lower shear forces, there is less tendency for the collected liquid to become an aerosol and to be removed from the collection tube with the existing gas. A similar effect is obtained by the proper angling the inlet transfer line relative to the collection tube wall. The closer the angle of the tube is to horizontal the lower the observed turbulence at the liquid surface. However, enough angle must be provided to ensure the majority of effluent is directed downward rather than upward on the liner wall. The two effects of back pressure and delivery angle combine to reduce aerosol formation in the collected liquid fraction. The success of optimizing these effects determines how close the inlet tube can come to the collection liquid, and thereby determining how high the liner may be filled before sample loss becomes a problem. When flow to the chamber is stopped, the chamber depressurizes. Once the sample chamber is depressurized, the liner may be removed by opening the top lid of the cassette.

[0042] The collection of fractions into disposable liners of collection chambers may be automated through the use of robotics. An automated system enables rapid substitution of test tube vials into and out of collection chambers and long unattended run times based on a quantity of vials available for substitution. A programmable robot automatically sequences cassettes between sample injections, thereby speeding up the process while reducing the margin for error. The automated system can collect on the order of thousands of fractions per month.

[0043] The automated system is contained in laboratory grade housing. The system is comprised of a robotic arm, a supply of test tube vials arranged upright in racks, and an automated version of a cassette assembly. In addition, the system may contain sufficient probes, valves and sample containers to achieve automated delivery of unfractioned samples into the chromatographic or extraction system.

[0044] The collection cassette and its automated mechanisms are designed for rapid sample collection and minimal stop time between chamber liner replacements. The cassette in the preferred embodiment has two banks of four collection chambers each. A lid is positioned above one bank of collection chambers in the cassette. The lid has four partially recessed annular bores corresponding to the four collection chambers in the cassette. The lid raises and lowers with action from pneumatic actuators mounted on the base of the housing and located an opposite longitudinal end of the lid. As the actuators simultaneously lower the lid onto the collection cassette, the top edge of each chamber engages the bottom edges of the lid corresponding to the rims of each partially recessed bore. The lid and chambers engage and form pressure tight seals in each chamber in preparation for sample fraction collection. The lid has transfer and waste line tubing passing through each recessed bore that correspond to each collection chamber. Each tubing pair enters a test tube as the lid is lowered onto the cassette. The spring wire attached to the inlet tubing guides an inlet tube into a test tube vial. An angled tip on the tube is forced against the inner wall of the test tube. As the lid has sealed on the row of collection chambers, a valve system dispenses the flow stream containing gaseous and liquid phases into the chamber liners from the sample fractionation process.

[0045] When all test tube vials in the pressurized cassette row have been filled and depressurized, the lids lifts off of the cassette. The cassette then moves laterally, or shuttles, until a row containing empty collection chamber liners is moved under the lid in place of the former row. The cassette is constrained to shuttle laterally along a path on the base of the housing. The lid lowers and engages the new row of chambers, thereby preparing the test tubes to accept sample fractions. Meanwhile, the former new row of chamber liner test tube vials containing liquid fractions are removed from the collection chambers and transported to open spaces in a storage tray via a robot arm.

[0046] In summary, samples in the preferred embodiment are dissolved in a minimum volume of modifier solvent and are collected in removable and reusable liners. Through controlling flowrate, velocity, temperature and pressure in the system, superior separation of near-supercritical elution fluid is obtained. Collection efficiencies of up to 98% of injected sample components may be realized. The cassette, by utilizing pressurized collection and cleaning solvent spent by the laboratory, which is economical and good for the environment. Laboratories and research facilities that demand purity of samples while maximizing output and minimizing waste will benefit from the proposed invention. Large-scale sample fractionation and collection, numbering in the thousands of samples per month, may be realized from the exemplary embodiment.

[0047] The method and its associated apparatus, described herein, provides for rapid separation and collection of volumes of the sample fractions from supercritical fluid chromatography systems such as those used in chiral separations. An exemplary embodiment is described using repetitive injections of higher volumes of samples into the system to effect rapid processing and recovery rates from similar elution components through a chromatography column. In contrast, analytical chromatography methods pertain to recovering multiple sample fractions that are separated and each gathered into different collection devices. The processing and separation method and apparatus for this exemplary embodiment is described in the specification.

BRIEF DESCRIPTION OF THE DRAWINGS

[0048] For a better understanding of the nature of the present invention, reference is had to the following figures and detailed description, wherein like elements are accorded like reference numerals, and wherein:

[0049]FIG. 1 illustrates a schematic flow diagram of the supercritical fluid chromatography system and the collection system including the sample cassette embodied in the invention.

[0050]FIG. 2 illustrates an exploded isometric view of a sample collection cassette.

[0051]FIG. 3 illustrates top and bottom plan views of the cassette lid.

[0052]FIG. 4 illustrates a plan view of an alternative exemplary embodiment of an automated fraction collection system.

[0053]FIG. 5 illustrates a side view of an alternative exemplary embodiment of an automated fraction collection system.

[0054]FIG. 6 illustrates an exploded isometric view of a shuttle sample collection cassette, lid, and mechanized controlled movement system.

[0055]FIG. 7 illustrates a detailed side view of the shuttle cassette and associated mechanical control apparatus.

[0056]FIGS. 8A and 8B illustrate detailed cross sectional views of transfer tubing before and after insertion into a test tube vial.

[0057]FIG. 9 illustrates an alternative embodiment of an integrated collection cassette having multiple rows of collection chambers.

[0058]FIG. 10 illustrates an additional alternative embodiment of a shuttle collection cassette for an automated system.

[0059]FIG. 11 is a schematic flow diagram of an additional exemplary embodiment of a collection system.

[0060]FIG. 12 is a side view of a collection system of an additional exemplary embodiment.

[0061]FIG. 13 is a graph illustrating detections of peak separations of a sample injection through a column.

[0062]FIG. 14 is a cross section of a collection container prior to insertion of transfer tubing.

[0063]FIG. 15 is a cross section of a collection container subsequent to insertion of transfer line.

DETAILED DESCRIPTION OF PREFERRED EXEMPLARY EMBODIMENTS

[0064] The preferred embodiment of the apparatus is illustrated in the flow chart of FIG. 1 within the perimeter line 10. Except where noted, specifications for a preferred exemplary embodiment are given for a system that accepts flows of 20 to 100 ml/min total flow (CO2 plus modifier flow) in the highly compressed state from the pumping system. Flowrates for alternative embodiments could range in orders of magnitude higher or lower through adjustment or substitution of system hardware and flow parameters.

[0065] In the preferred exemplary embodiment, the SFC collection system is composed of a moderately restrictive, thermally regulated transfer tube 12 which extends from a back pressure regulator 14 into a multi-port distribution valve 22 and from the valve to a variety of flow paths leading either through discrete collection chambers 32 or directly connected to a vented common waste container 26.

[0066] Expanded elution fluid leaves the backpressure regulator 14 at a velocity of approximately two to five times the flow velocity upstream of the backpressure regulator 14 and under backpressure of approximately twenty to forty bars. Variations in the expansion occur as a result of the changing modifier solvent concentration from 2.5 to 50 percent over the course of a separation.

[0067] Initial separation of the liquid phased sample from carbon dioxide gas occurs immediately at the point of initial decompression within the backpressure regulator 14 of the SFC or SFE system. By providing downstream restriction, a minimum backpressure sufficient to prevent the formation of solid CO2 can be maintained while liquid CO2 is present in the transfer lines 12. The degree of CO2 evaporation is a function of both the available heat transfer in this region and the downstream flow restriction which limits the amount of expansion available to the decompressing fluid. Due to the pressure drop across the backpressure regulator 14, a fraction of the emerging CO2 will evaporate, typically causing a significant drop in the temperature of the emerging fluid.

[0068] Further separation and evaporation of CO2 from the organic modifier occurs in stainless steel transfer tubing 12 running between the first backpressure regulator 14 and the cassette 24. The transfer tubing 12 containing a flow stream of the biphasic CO2 and modifier is exposed to a series of heaters 16, 18 designed to optimize thermal transfer to the biphasic fluid in the flow stream. Ideally, this heater series transfers sufficient energy to the liquid CO2 portion of the emerging fluid to allow for complete evaporation of the liquid CO2 and raises the fluid temperature sufficiently to prevent ice from forming externally on the transfer tubing 12.

[0069] During the CO2 evaporation process within the first heated zone, significant separation between the gaseous CO2 and liquid modifier occurs. However, the separation to pure CO2 and pure organic modifier is never realized. Some organic modifier is typically evaporated into the gas state. The degree of evaporation is largely dependent on the absolute temperature of the fluids within the transfer tubing 12. While organic modifier evaporation does lead to lower recovery of liquid phase when it reaches the collection cassette 24, it does not necessarily reduce the recovery of dissolved components of interest which do not typically have low enough boiling points to convert to vapor. A fraction of CO2 will also remain dissolved in the organic liquid modifier. Both temperature and pressure determine the amount of residual CO2. Higher temperatures reduce CO2 solubility while higher pressures increase CO2 solubility. Turbulent flow of the CO2 gas within the narrow tubing also produces a strong shearing force that propels the liquid down the walls of the transfer tube 12. This very turbulent flow frequently causes small droplets at the liquid surface to rip away from the bulk liquid and become entrained into the rapidly moving gas phase of the fluid down the transfer tube 12. Such an effort is called aerosol formation, or “misting.”

[0070] A plurality of heaters may be mounted in series to heat t the elution fluid. In FIG. 1, the preferred exemplary embodiment has an evaporator heater 18 and a trim heater 20 mounted in series after the backpressure regulator 14. The evaporator 18 is heated with an appropriately sized cartridge heater and controlled by an appropriate heater controller. In the preferred embodiment, transfer tubing 12 is tightly coiled around the heating assembly and optimized for thermal contact. The elution fluid is heated to within the control temperature of the evaporator 18, which is between approximately 5 to 50 degrees C., to protect heat sensitive compounds from being damaged. The objective is to boil CO2 out of the elution fluid as the fluid passes through the evaporator 18. To complete the required heat transfer, biphasic elution fluid inside transfer tubing 12 enters the final heat exchanger, which is a trim heater 20. In the preferred embodiment, the trim heater setting is typically above the evaporator 18 setpoint. The heater 20 is used not only to suppress aerosol formation within the transfer tube 12 but also to control the level of dissolved CO2 in the liquid phase.

[0071] It is beneficial to slow the velocity of fluids within the transfer tubing 12 passing through the heater series 18, 20. The fluid velocity is slowed inside the transfer tubing by 12 placing a restrictive orifice or smaller diameter tube immediately downstream from first heater series. Elution fluid exits the evaporator 18 and enters a flow restrictor 16, which provides a higher backpressure in the evaporator 18 and thereby slows the flow and increases the contact time of the liquid CO2 phase. The restrictor 16 also insures a high enough backpressure to prevent the liquid carbon dioxide from forming solid carbon dioxide, also known as dry ice. The restriction increases the backpressure in the heated zone and reduces the amount of gas expansion. In an alternative exemplary embodiment, the velocity of fluids can be slowed after all heaters, however, such a configuration does not control the final expansion of CO2 which can result in uncontrolled cooling of fluids within the transfer lines. As a result, the ability to actively suppress aerosol formation may be diminished.

[0072] After existing the trim heater 20, transfer tubing 12 connects to the common port of a valve system 22. The valve system in the preferred exemplary embodiment is a multi-port sector valve 22. As elution fluid from the peak of interest passes through the valve system 22, the gas and liquid phases are directed into either a collection cassette 24 or to a waste stream container 26. The outlet ports on a multi-port selection valve 22 are connected to a plurality of transfer tubing lines 28. The transfer lines 28 pass through a cassette lid 30 and into discrete chambers 32 within the cassette 24. The transfer lines 28 have airtight and pressure resistant connections into and out of the cassette lid 30. The remaining ports in a multi-port selection valve 22 connect to waste transfer lines 34. In an alternative exemplary embodiment, multiple discrete valves are installed and connected to the incoming transfer line 12, having each valve port connected to an individual collection chamber 32 in the cassette 24 and a discrete valve connected to a waste line 34.

[0073] Inlet lines 28 entering a collection chamber 32 insert into a test tube vial 36 within a chamber 32. Liquid phase 38 is captured in a test tube 36 while gaseous phase escapes out of a chamber 32 through a discharge line 40. Gas in the discharge line 40 is flowing at high pressure. Discharge lines 40 from the cassette 24 run through a pressure relief switch 42 to protect the cassette and upstream components from possible damage due to over-pressurization from a system malfunction.

[0074] Referring additionally to FIG. 2, a preferred exemplary embodiment of the cassette 24 comprises four discrete collection chambers 32. However, in alternative embodiments, one or more individual chambers 32 are possible in the cassette 24. Each collection chamber 32 in the preferred embodiment is a closed system that is the final separation point of liquid and gaseous phases. Chambers 32 are hollow cylinders constructed of high strength transparent plastic to allow visual monitoring of separation and collection processes. The cassette chambers 32 can be formed of stainless steel or other appropriate laboratory-grade materials. The chambers 32 sit parallel and upright in the cassette 24. Each chamber 32 is constrained at its upper at its upper and lower ends within a molded frame 44, 46. Each chamber 32 is set with the open end surrounded by the upper molded frame 44 and the lower end partially embedded into the lower molded frame 46. Communication of liquid or gaseous phases between chambers 32 is prohibited by seals 48 that are seated in a groove 50 at the top, open end of each chamber 32.

[0075] Each collection chamber 32 houses a removable, replaceable liner. A standard glass test tube vial 36 functions as a liner and is seated upright inside each the chamber 32. The closed bottom of a test tube vial 36 rests on the base of the chamber 32 and is easily removable. Once inserted, the top of the test tube vial 36 must be lower than the combined height of a chamber 32 and the internal recessed bore 60 (FIG. 3) of a lid piece 30 when the lid and cassette 24 are engaged. A test tube vial 36 and a chamber 32 are a single pressurized system that communicate through the top of the chamber 32. The test tube vial 36 functions as a disposable liner for the chamber 32 to capture the liquid phase 38 that has separated from the flow stream. The inside of the vial 36 and the annular space of chamber 32 surrounding the vial are equiliberated to the same pressure, which is a range of approximately 20 to 100 psig during separation processes for a flow stream up to 50 ml/min. This arrangement enables sample fraction collection at high pressure using standard laboratory glass test tube vials 36 without a risk of breaking the glass vial inside the chamber 32.

[0076]FIG. 2 illustrates the cassette 24, comprising a rectangular frame securing four upright chambers 32. The upper section 44 and lower section 46 of the molded frame hold the chambers 32 in place. The frame is completed by two rigid rectangular end pieces 52 attached to the upper and lower sections. Each end piece 52 is a metal plate fastened to the upper 44 and lower 46 frame sections with machine screws 54. Butterfly latches 56 are installed at te top of both rigid end pieces 52 secure the lid piece 30 to the top of the cassette 26. The lid 30 may be removed manually between sample injections for quick access to, and removal of, chamber liners 36. As illustrated in FIG. 1, the bottom of each chamber 32 has a transfer tube or orifice 33 running completely through the base of a collection chamber and lower frame 46. The orifice 33 through the chamber base 46 can be used to remove liquid phase fluid from a chamber 32 without opening the chamber or depressurizing the chamber 32. The sample discharge port 33 also permits easier drainage and cleaning of the chamber 32 during maintenance of the cassette 24.

[0077]FIGS. 3A and 3B illustrate top and bottom views of the removable cassette lid 30, respectively. The lid 30 has four sets of three boreholes 58 in a triangulated pattern positioned such that each set of boreholes is directly over each of the chambers 32 when the lid 30 is engaged to the cassette 24. The bottom face of the lid 30 has partially recessed bores 60 positioned directly above each chamber 32 when the lid 30 and cassette 24 are engaged. The diameter of a recessed bore 60 is sized slightly smaller than chamber 32 diameters. The recessed bore's 60 perimeter is positioned completely inside of a seal 48 when the lid 30 is fastened to the cassette, as illustrated in FIG. 2. The recessed boreholes 60 allows a test tube 36 to stand taller than the top planar surface of the upper frame section 44 of the cassette 24 so that a test tube 36 may be removed without reaching into a collection chamber 32, thereby possibly cross-contaminating subsequent samples. To guide the lid 30 and cassette base 24 together when engaging, alignment pins 62, illustrated in FIG. 3, are formed on the outer, top surface of the cassette frame 24. Partially recessed bores 63 in the lid 30 receive the alignment pins 62 from the cassette frame 24. Catches 64 for the butterfly latches 56 are attached to each long end of the lid 30.

[0078] Inlet transfer tubing 28 carries liquid and gaseous phases into test tube vials 36 housed in each collection chamber 32 of the cassette. Each inlet tube 28 fits through a hole 58 in the lid 30 and inserts into a test tube vial 36. Proper fittings on the tubing 28 provide airtight connections that can also withstand pressure forces in the SFC system. Inlet tubing probes 66 direct elution fluid into a test tube vial 36 and an outlet tube 68 provides an escape route for gas that is under pressure to exit the chamber 32 and discharge to waste collection 26.

[0079] In the preferred embodiment, fractions are collected in one chamber 32 of the cassette 24 at a time. During the fractionation process, both the liquid phase and the gas phase discharge into the collection vial 36 where final separation takes place. The pressurization of the collection chamber 32 serves to slow down the velocity the CO2 within the chamber 32. This in turn reduces the magnitude of shear forces occurring between the CO2 gas and the collected liquid at the bottom of the liner 36. With lower shear forces, there is less tendency for the collected liquid to become an aerosol and removed from the collection liner 36 with the existing gas. A similar effect is obtained by the proper angling the inlet transfer line relative tot he collection liner 36 wall. The closer the angle of the tube 66 is to horizontal the lower the observed turbulence at the liquid surface. However, enough angle must be provided to insure the majority of effluent is directed downward rather than upward on the liner 36 wall.

[0080] The biphasic elution fluid enters a chamber 32 via a transfer line 28 from the valve system 22. As illustrated in FIGS. 8A and 8B, the tip of the transfer tube 66 is a probe preferentially positioned tangential to the inner wall of the collection vial 36 and with a slight downward angle, usually less than 45 degrees from horizontal. Attached to the probe 66 is a guiding spring wire 70. The spring wire 70 is bowed away from the probe 66. The spring wire 70 acts as a guide for the probe 66 as the probe descends into a test tube vial 36. When the probe 66 is properly inserted into a test tube vial 36, the bowed section of the spring wire 70 contacts the circumferential edge of the open end of a test tube vial 36. As the tubing 66 continues into the test tube vial 36, the spring wire 70 compresses against the inner surface of the vial 36 and pushes the probe 66 towards the opposite side of the vial 36. As a result, the angled tip of the probe 66 is pressed against the inner wall of the test tube vial 36.

[0081] The spring wire 70 is extruded from inert materials that will not chemically interfere with collected samples in the test tube vials 36. In an alternative exemplary embodiment, the probe section 66 of the transfer tubing 28 is a rigidly held stainless steel probe attached to the cassette lid 30. Metal versions of probe 66 may be terminated with a larger OD Teflon tube sleeved onto the metal probe to prevent scratching and possible rupture of the inner wall of the collection liner 36.

[0082] Both the organic liquid and CO2 gas follows a descending spiral path along the inner wall to the bottom of the collection liner 36. The liquid phase collects at this point and begins to fill the test tube vial 36. The CO2 gas continues in a path up the center of the vial 36 to a vent through the top of the collection chamber 32. A restrictive transfer line attached 72 to the vent causes the CO2 gas to pressurize the collection chamber 32 both inside and surrounding the collection line 36. The degree of back pressurization within the chamber is roughly proportional to the composition of CO2 in the original mobile phase.

[0083] The two effects of back pressure and delivery angle combine to reduce aerosol formation in the collected liquid fraction. The success of optimizing these effects determines how close the inlet tube 66 can come to the collection liquid, and thereby determining how high the liners 36 may fill before sample loss becomes a problem. When flow to the chamber 32 is stopped, the chamber depressurizes. Once a chamber is de-pressurized, the test tube vial 36 containing liquid phase may be removed by opening the top lid 30 of the cassette 24.

[0084] The outlet line tubing 72 from each chamber 32 is connected to a fixed restrictor 42 to keep pressure inside the chambers 32. The fixed restrictor 42 raises the upstream pressure between approximately 20 and 100 psig depending on CO2 flow rate. Each discharge line 72 passes through a pressure switch 78 to protect against overpressuring and rupturing. Pressure in each chamber is monitored visually with a pressure gauge 76 that is threaded into the lid 58 over each chamber 32. Discharge lines 72 are directed to a waste collection tank 26, from which the CO2 is vented. To increase laboratory safety, the system should not have any exposure of waste effluent, samples, or vented CO2 to ambient laboratory air. The liquids and gasses in the system remain in a contained system that can be directed to a hood or safety exhaust 26 to maximize safety for the technician.

[0085] The volume of the captured fractionated liquid phase 38 in the collection vial 36 is controlled manually or automatically. Automatic control in the preferred exemplary embodiment of the valve system 22 and is comprised of one or more valves and an electronic controller. The valve system 22 is designed to offer rapid responses to a manual or automated start/stop signal. A signal can result from detection of a component of interest emerging from the high pressure flow system. A start signal would be generated at the initial detection of the component while a stop signal would be generated at the initial detection of the component while a stop signal would be generated at the loss of detection. The effect of the stop signal is to divert the flow to waste lines 26 or to another chamber 32. An alternative embodiment of a type of start/stop signal may be based on a time-table rather than physical detection of components. The controller may also have features to limit the access time or flow volume allowed to an individual chamber 32. In addition, the controller may allow or prevent the system from cycling back to the original chamber 32 if more fractions are desired than there exist available collection chambers 32.

[0086] An alternative exemplary embodiment of the collection cassette and system is illustrated in FIGS. 4 through 7. This embodiment is an automated system that utilizes a robotic arm 80 to replace chamber collection liners 36 after filling with sample fractions. The robotically controlled unit is designed for rapid filling and replacement of chamber liners 36 combined with a long unattended run time. Supply trays 86 of clean test tube vials 36 that function as chamber liners 36 are located within the unit's housing 82. A robotic arm 80 is controlled to replace one or more liners 36 from a row of collection chambers 32 in a collection cassette 84 with liners 36 from a fresh supply rack 86. The robotic arm 80 is mechanized to replace liners 36 on a first row of the cassette 84 while liners 36 on a second row are automatically moved into place. This robotically automated alternative embodiment provides faster sample collection through a minimum of down time to replace liners 36 as well as the ability to collect a greater number of samples during the unattended session.

[0087]FIGS. 4 and 5 illustrate the plan and side views, respectively, of an automated alternative exemplary embodiment of the SFC sample collection system. The components for the system are partially enclosed with a laboratory-grade housing structure 82 having a raised mounting base 88 within the housing 82. The housing 82 is supported with adjustable feet 90 that are distributed around the base of the housing 82. The feet 90 adjust the level the housing 82 to compensate for uneven or slanted surfaces. Supplies of uncontaminated test tube vials 36 are stored in racks 86 placed on a raised interior base 88 of the housing 82. Each test tube vial 36 is held upright and secure in-place in a rack 86 by molded supports. Each support rack 86 is held upright and secured in-place in a rack 86 by molded supports. Each support rack 86 consists of circular sections attached tangentially to neighboring sections, forming multiple rows and columns. The molded supports loosely secure test tube vials 36 that are held in each circular opening of the racks 86. The vials 36 are maintained equidistant from each neighboring vial to provide adequate spacing for a grabbing jaw 92 on a robotic arm 80 to grasp a vial 36 without interference from a neighboring vial. The spacing also prevents chipping or breakage during movement and replacement of the rack 86. Two racks 86 of test tube vials 36 are illustrated in the Figures, however the system could easily expand to a plurality of racks of the vials 36.

[0088] An alternative exemplary embodiment of a cassette 84 and associated system devices is installed on the raised interior base 88. The cassette 84 has a plurality of rows and chambers that are constrained to lateral movements that are automatically controlled with a pneumatic actuator 96. This cassette 84 9s termed the “shuttle cassette,” or simply “the shuttle.” FIGS. 6 and 7 illustrate the shuttle cassette 84 in isometric and side views, respectively. The shuttle cassette 84 is constructed similar to the exemplary embodiment with an added row of collection chambers 102. The shuttle 84 comprises upper and lower rectangular molded frames 98, 100 supporting a plurality of rows of upright cylindrical collection chambers 102. The shuttle 84 is constructed with two rows of four cylindrical collection chambers 102 in each row. The size of the shuttle 82 can be modified to add additional rows of chambers 102 or additional chambers per row, such as an alternative embodiment featuring three rows of chambers 102 illustrated in FIG. 10. The shuttle cassette 84 is formed on two opposite ends with ridge rectangular plates 104. Each end plate 104 is fastened to the upper 98 and lower 100 molded frame sections with machine screws 106. The shuttle 84 may be constructed with permanent attachments and fittings, however, a shuttle that readily disassembles allows easier and thorough cleaning and replacement of worn or damaged components.

[0089] The collection chambers 102 are formed of high-strength transparent plastic, which allows visual monitoring of the collection process inside of each chamber 102. As an alternative, the chambers 102 may be formed of stainless steel or a similar high-strength material compatible with SFC parameters described herein. Each cylindrical chamber 102 is set into the lower molded frame 100 for base support. The upper molded frame section 98 is secured near the open, top end of each chamber 102. Each chamber 102 extends above the top surface of the shuttle 84 at a standardized distance adequate to seal the chambers 102 with an automated lid piece 108. Standard laboratory test tube vials 36 may be inserted into each of the chambers 102 to act as a removable or disposable liner for each chamber.

[0090] The automated shuttle cassette 84 is constrained to lateral movements on the inner raised base 88. The lower molded frame section 100, or base, of the shuttle cassette 84 has a horizontally bored hole 110, illustrated in FIG. 7, running perpendicular to the open sides of the shuttle. Offset from the shuttle 84 is an actuator 96 installed on the raised base 88 of the housing unit 82. Attached to the actuator 96 is a rod 94 or controller arm. The rod 94 is constructed of a rigid material, such as stainless steel, and inserts into the bored hole 110 in the base of the shuttle cassette 84, wherein it is firmly attached to the base frame 100. The actuator 96 executes lateral movements of the shuttle 84 according to commands sent from the programmable control system. In an alternative embodiment, the base of the shuttle 100 has small rollers 112 installed around the base, as illustrated on FIG. 7. The rollers 112 are guided laterally by grooved tracks in the base of the housing 88. The tracks not only constrain the movement of the shuttle 84 but also remove tension from the controller arm 94 and actuator 96 gears caused by the shuttle 84 drifting into angled movements caused by uneven friction on the rollers, initial off-center displacement after shuttle 84 installation, or irregularities on the surface of the housing base 88. Other methods of providing constrained lateral movement are possible in alternative embodiments, such as utilizing guide tracks wherein guides on the shuttle 84 are enclosed within tracks riding on ball bearings.

[0091] Referring to FIGS. 6 and 7, the lid 108 of the shuttle cassette 84 is automatically controlled to engage a row of collection chambers 102 after the shuttle is moved into place directly below the lid 108 by the lateral actuator 96. In the alternative embodiment, the lid 108 is constructed of stainless steel. However, high-density plastic, or a similar material having equivalent rigidity and composition for use in the collection system, is sufficient. The lid 108 has a hole 114 through each longitudinal end, bored parallel to the vertical axis of the lid. The holes 114 in each end of the lid 108 are sized to fit a threaded rod 116. Two nuts 118 threatened above and below the lid 108 secure the lid to each rod 116. The lid 108 is constrained to move only in the vertical plane. The movements of each rod 116 are controlled by actuators 120 mounted to the raised base of the housing 88. The two pneumatic actuators 120 controlling the lid movements are synchronized to move the rods 116 vertically, thereby raising and lowering the lid 108 onto a row of collection chambers 102 in the shuttle cassette 84.

[0092]FIG. 7 illustrates the lid piece 108 raised above the shuttle 84 prior to engagement. The bottom face of the lid 108 has four bores 122 partially recessed into the lid corresponding to four chambers 102 in a row of the shuttle. As the lid 108 is lowered by the pneumatic actuators onto the shuttle 84, each chamber 102 of a row partially inserts into a recessed borehole 122. The lid 108 stops at a programmed point at which the circular edge of each bore 122 engages and seals against the flat upper surface of the shuttle frame 98. Each partially recessed borehole 122 in the lid 108 has a diameter larger than the chambers 102 diameter. As the lid 108 lowers onto the shuttle 84, the recessed boreholes 122 are lined up with the top, open end of each chamber 102. The larger diameter recessed boreholes 123 each totally enclose the open end of each chamber 102. An appropriate sealing O-ring or similar component is placed around the top of each chamber 102, between the top of the shuttle 84 and the lid 108, to provide an airtight and pressure resistant seal when the two components engage. Alignment pins 124 are located on the top surface 98 of a shuttle 84 at both ends of each row of chambers 102. The pins 124 are shaped as half-spheres on the top surface of the shuttle 84 and provide additional protection for shuttle collection chambers 102 from misalignment of the shuttle 84 to the lid 108. As the lid 108 engages onto the shuttle 84, the alignment pins engage corresponding bores 126 in the lid.

[0093] A collection chamber 102 is a discrete system that is the final separation point of liquid and gaseous phases. Communication of liquid or gaseous phases between chambers 102 is prohibited through the lid 108 that seals each chamber airtight as it automatically lowers onto a row of chambers in the shuttle cassette 84. Similar to the exemplary embodiment of the cassette, each chamber 102 in the shuttle 84 holds a chamber liner 36 to catch fractional liquid phase. The liner 36 is a standard laboratory test tube vial 36. The closed bottom of the test tube 36 rests at the base of each chamber 102, which rests on the lower molded frame of a shuttle 100. A test tube vial 36 and chamber 102 communicate as a single pressurized system. FIG. 8B illustrates the position of the open end of a vertically disposed test tube vial 36 below the top of a recessed borehole 122 after the lid 108 engages the shuttle 84. The inner pressure of the test tube vial 36 and the chambers 102 annular space surrounding the vial are equilibrated and range from approximately 20 to 100 psig during collection processes. This arrangement enables sample fraction collection at high pressure using standard lower pressure glass or plastic vials by equilibrating the pressure forces inside and outside the vial 36.

[0094] As illustrated in FIG. 7, the lower, closed end of each chamber 102 has a sample discharge port 128 running completely through the lower shuttle frame 100. A plug is inserted into each sample discharge port 128 during regular use of the shuttle 84. The sample discharge port 128 permits removal of liquid phase that is collected directly into a chamber 102 without using a liner. By withdrawing liquid phase through the sample discharge port 128, the liquid phase may be collected without disengaging the lid 104 from the shuttle 84. Liquid phase may be evacuated from a chamber 102 under pressure or gravity fed out of a chamber after chamber depressurization.

[0095] Inlet 66 and outlet 68 tubing for transferring influent and effluent liquid and gas phases between the shuttle cassette 84 and external transfer lines are illustrated in FIGS. 6 and 7. Inlet 66 and outlet 68 tubing for the shuttle 84 pass through the lid 108. Transfer tubing 66, 68 is constructed from high-pressure stainless steel or equivalent materials. Inlet tubes 66 carry gaseous and liquid phases into a collection chamber 102 under high pressure. Outlet tubes 68 carry separated gaseous phase to a waste tank 26 for venting or disposal. The lid section 108 has four sets of three holes 134 in triangular formations that pass through the lid and are located to correspond with collection chambers 102 when the lid is engaged to the shuttle cassette 84.

[0096] In addition to transfer tubing, one of the holes 134 permits measurement of pressure forces inside a chamber with a pressure gauge 76 threaded into the hole 134 from top of the lid 108. The transfer tubing 66, 68 and pressure gauge 136 all have pressure resistant airtight fittings specified to withstand pressure forces created in the SFC system. Transfer tubes 66, 68 installed below the lid 108 insert into a test tube vial 36 when the lid 108 is engaged to the shuttle cassette 84. The tip of each inlet tube 66, or probe, is constrained to an angle less than 45 degrees and wrapped with non-reactive spring wire 70 that is bowed along the vertical section, similar in construction and purpose as described in the preferred embodiment. The spring wire 70 serves to angle the inlet tubing 66 inside a test tube vial 36 by applying pressure forces against the vial's 36 inner wall. As a result, the open tip of the inlet tube 66 is forced tangentially against an opposing inner wall of the vial 36. This configuration of the inlet tube 66 is desirable because it causes the liquid phase that exits the inlet tube 66 to contact a side wall of the vial 36 and swirl down the inner wall of the vial 36 in a spiraling motion. The swirling action provides the final separation process of liquid phase from entrained gaseous phase while preventing re-entrainment and loss of sample fractions from the liquid phases into gaseous phases or aerosol mists that can be carried away with gaseous phases to a waste vent 26.

[0097] In an alternative exemplary embodiment, a robotic arm, such as a Cartesian or three-dimensional robotic arm, is programmably controlled to move test tube vials between supply racks and the shuttle cassette collection chambers. FIGS. 4 and 5 illustrate a three-dimensional robotic arm 80 mounted to a wall of the unit housing 82 near the shuttle cassette 84. A host PC or microcontroller issues positioning commands for the arm's movement and controls automated functions. The arm 80 has a jaw 92 to grab and place test tube vials 36 into the shuttle cassette 84 from the test tube supply racks 86. The jaw 92 is controlled to grip test tube vials 36 of specific outer diameter and at specific locations within the unit 82. In the alternative embodiment illustrated in FIG. 5, the robotic arm 80 is gripping one test tube 36 in its jaw 92 to move the test tube between the shuttle 84 and a supply rack 86. To increase the volume of vials 36 exchanged, the gripper jaw 92 could be modified to grip two or more test tube vials, multiple jaws could be placed on a single arm 80, or multiple robotic arms could work on the same embodiment. The arm 80 acts in concert with the automated movements of the shuttle 84. As a row of chambers 102 in the shuttle 84 is engaged to the lid 108, the robotic arm 80 replaces test tube vials 36 in the shuttle that are filled with collected sample fractions with fresh vials 26 from a supply rack 86. When a row of test tubes 36 in the shuttle 84 have been replaced, and the row of vials 36 under the lid 108 have captured liquid phase fractions, a programmable controller signals the pneumatic actuators controlling the lid 120 to disengage and move the lid 108 away from the shuttle 84. The lateral control 96 of the shuttle 84 is then signaled to move the shuttle such that the rows of chambers 102 containing clean, uncontaminated test tube vials 36 correspond to a position underneath the lid 108 prior to engagement. The lid actuators 120 are then signaled to engage the lid 108 again to the shuttle 84, thereby preparing the chambers to receive liquid phase fractions. The robotic arm 80 next grabs vials 36 from the exposed shuttle chambers 102 that contain liquid phase fractions and places them into a supply rack 86. The arm 80 then replaces an uncontaminated vial 36 into each empty chamber 102 until a row of chambers is completely filled with fresh test tubes. This process is repeated for the length of a sample run or until the system is depleted of uncontaminated test tube vials from the supply racks 86.

[0098] An alternative embodiment of a collection cassette is illustrated in FIG. 9. An integrated cassette 140 consists of multiple rows of wells 144 in a grid pattern formed similar to a titration tray. The smaller footprint of the integrated cassette 140 can increase the density of collection chambers over the shuttle cassette 84. The integrated cassette 140 also functions as a storage tray for gathered liquid phase fractions. Therefore, time and expense are saved during sampling procedures by removing the steps of the substituting chamber liners 36 and replacing liners from a separate storage area. By modifying the lid 108 and mechanics of the automated collection system, the integrated cassette 140 may serve as its own sample collection cassette and storage tray and can rapidly receive fractions without having to replace liners 36 between each sample injection. The robotic arm 80 in the system may replace integrated cassette 140 units as a whole after a sampling event is completed or chamber wells 144 contain the desired amount of liquid phrase fractions. A plurality of integrated cassettes 140 are stored in the automated collection system providing the means for hundreds of collected fractions during an automated run. A preferred construction of an integrated cassette is a 4×6 chamber array in the deep-well micro titer plate format used commonly in the pharmaceutical industry. Such a format improves automation storage density not only due to more chambers per area, but these chambers are also easily stackable, which gives an added dimension of sample storage capacity. This alternative embodiment is a shuttle cassette tray 140 formed from high-strength materials such as plastic, resin, or stainless steel.

[0099] The integrated cassette tray 140 is also advantageous for rapid fraction collection because it can be modified to contain replaceable liners 36 in the wells 144 or use no liners, thereby collecting liquid fractions directly into the wells 144. The integrated cassette 140 can be replaced as a unit after wells 144 are filled with liquid phase fractions.

[0100] An alternative embodiment of an automated system using a cassette tray would appear similar to that illustrated in FIG. 4 but with certain modifications. Modifications to the automated system include spacing for a supply of cassette trays 40 instead of test tube racks 86, sizing of the lid piece 108 and associated mechanized controllers 120 and transfer tubing 66, 68, sizing of lateral mechanized controllers 96 for the tray 140 while switching between rows of chambers 144 during fraction collections, and modification of a robotic arm 80 to no substitute filled cassette trays 140 with new trays from a supply area. An alternative to this configuration is having a moveable lid section 108 connected to a robotic arm 80 that engages each flow of chambers in a supply rack of trays 140 without ever moving the trays.

[0101] As can be understood from the above description, the sample collection system has several advantages, for example: it provides simplified pre-SFC sample collection; it collects only fractions of interest from the injected sample; it collects purified samples into removable, inexpensive, and disposable collection vials; it provides extremely efficient and controllable gas and liquid phase separation, thereby providing up to 98% consistent sample recovery; it is environmentally friendly and economical because it eliminates additional use of solvents to collect, trap, or recover samples, and clean unnecessary associated mechanical separation equipment; it allows high speed, high volume, and high purity SFC sample collection.

[0102] An additional alternative embodiment of an SFC collection system is illustrated in the flow diagram of FIG. 11. The embodiment collects liquid phase fractions from an SFC system without using a pressure scheme in the collection vessel. Instead of expanding and separating the mobile phase flow in a collection chamber under a pressure force, as described in reference to the collection system in FIG. 1, an expansion space is provided in the flow stream prior to flow entering a collection vessel to slow the mobile phase flow stream and allow gasses to expand out of the liquid phase. By slowing the speed of the mobile phase flow stream through an expansion space and allowing the flow to expand, gasses are separated from the liquids prior to entering a liquid collector. As a result, the flow does not re-aerosolize or disturb liquid being collected in a collection vessel. Therefore, when mobile phase reaches a collection vessel, the flow is substantially separated into liquid and gaseous components. No pressure scheme is applied in the collection vessel since the flow expansion space slows the linear velocity and significantly reduces pressure of the flow stream.

[0103] In an exemplary embodiment, the expansion space is provided by using large bore diameter tubing 300 between distribution valve 22 to a collection vessel 302. For example, in FIGS. 1 and 2 instead of {fraction (1/16)} inch diameter tubing 22 between valve 22 and a cassette 24, ⅛ inch tubing is provided between a valve 22 and a collection vessel 302. Assuming a pressure of 60 psi inside transfer tube 22, a four-fold increase in cross-sectional area in the flow path would induce an approximate four-fold reduction in linear velocity of the flow stream. As one skilled in the art will observe, the sizes shown are exemplary. Exact sizes, shapes, flow stream measurements, and structures of may vary depending on linear velocity, volumetric flow rate, configuration and size of the system assembly, and any other factors necessary for an SFC system design and operation. For example, substitutions for a larger diameter tube upstream of a collection vessel could be a rigid column, pipe, or chamber device having a flow path large enough to provide adequate expansion of the mobile phase and reduction of linear velocity of the flow stream.

[0104] As described previously in reference to FIG. 1, an exemplary collection system using a cassette 24, the collection chamber 32 served as a broad point in the flow path wherein mobile phase flow could slow and expand. Liquid phase flow is collected under a pressure scheme to prevent the formation of aerosols and loss of sample fractions. Linear velocity of the mobile phase flow is high within the transfer lines 28, which have a small diameter, such as {fraction (1/16)} inch, and is subjected to high pressures associated with the SFC process. A smaller diameter tube 66 is also easier to receive into a test tube as opposed to receiving large bore diameter tubing.

[0105] Exposing the mobile phase flow stream to a larger-diameter flow path slows the linear velocity of the mobile phase. Slowing the mobile phase reduces the magnitude of shear forces occurring between mobile stream gasses and the liquid flow stream in transfer lines leading to a collection vessel. By lowering shear forces in the mobile phase, there is less tendency for the stripping of liquid fractions into mobile phase gasses, thereby creating an aerosol. Stripping forces cause aerosols, containing sample fractions, to escape collection and proceed into a waste discharge collector, thereby losing valuable volumes of eluted samples.

[0106] Because separation of the flow stream is performed in transfer line 300 or other expansion space in the collection system that is upstream of a liquid fraction collection point, any number of collection devices 302 can be of used having varying sizes and shapes. The flow diagram of FIG. 12 illustrates how four collection devices 302 are connected to distribution valve 22. Using a plurality of devices 302, the system has greater liquid phase retention capability which translates into longer unattended run times. Because separation of gasses from the mobile phase occurs quickly and simply upstream 300 of the collection devices 302, the large volumetric capacity of liquid phase retention in the present invention removes time-consuming bottlenecks caused by additional processes typically found in SFC sample solute collection processes and promotes rapid sample injection, elution, and collection.

[0107]FIG. 13 illustrates four discrete, upright collection bottles 310 that serve as collection devices inside of safety housing312. Bottles 310 are laboratory-grade one-liter bottles, however, any container sized appropriately for the process flow and having sufficient strength and chemical compatibility to provide a collection vessel for liquid phase flow can function to retain liquid phase sample fractions in the present invention. Bottles 310 are constructed of glass, plastic, or any laboratory-grade material compatible with samples and substances in the SFC system. Bottles 310 have high volumetric capacity and therefore allow for longer unattended runs of the SFC system and easy removal, transport, and storage. In addition, many standard laboratory bottles can function as collection containers, thereby significantly reducing costs of specially-made or adapted liquid fraction collection devices.

[0108] Exhaust gasses, such as CO2, emanating from the phase-separated mobile phase flow stream are discharged from bottles 310 through flow lines 304 to waste collection system 306. As a result of the process, the collection vessel is mildly pressurized. The pressurization is useful for safety and waste flow purposes. For example, a bottle may be pressurized at three to five psi to contain modifier solvent gasses and control the flow of gasses into a waste line 304.

[0109] Referring to FIG. 13, bottles 310 are contained in a safety housing 312 that is rectangular in shape with rigid, planar opposing ends 314 attached to top 316, bottom 318, and back 320 ends. Housing 312 has a front access panel 322 constructed of transparent material permitting viewing of internal components and processes. Access panel 322 can be removed for access to, and removal of, bottles 310. A plurality of holes324 receive flow lines 300, 304. Flow line 304 provides an escape route for gasses and vapors to exit under pressure and discharge to waste collection system 306. A gauge 326 is attached to the top-end 179 and displays internal pressure.

[0110] A collection device with large volumetric capacity also provides greater internal space for dissipation of pressure spikes while lowering the dangers of cracking or exploding the device. For safety purposes, any device used in the present invention for collection of the liquid fractions should have adequate strength to withstand potentially dangerous pressure fluctuations associated with the SFC process. If breakage of a collection bottle 310 does occur, housing 312 can contain shrapnel and hazardous substances from exploding into a laboratory area. For additional safety, glass bottles may also be treated with a shatter-resistant coating.

[0111] The alternative embodiment of the present invention has an advantage of allowing unattended runs that can be combined with a rapid injection process to provide a high volume of collection capacity without renewing the collection system with empty collection devices. Although FIG. 13 depicts four bottles 310, only one collection device is necessary, as depicted with device 302 in FIG. 11. Each collection device operates independent of one another. The system can use inexpensive, off-the-shelf laboratory items, is self-contained, and requires no additional support structure for a collection vessel. As one skilled in the art will recognize, the combination of a bottle and associated tubing illustrated in FIG. 13 is an exemplary embodiment for the present invention. Any type of vessel or container space that is compatible with samples and fluids used in the process described herein can be used with the present invention to collect liquid sample fractions.

[0112]FIG. 14 depicts flow lines 300 in and 304 out of bottle 310 and FIG. 15 depicts a similar view having line 300, 304 received inside a bottle 310. To promote separation of phases and a gentle flow stream delivery into bottles 310, tip 328 of flow line 300 is angled away from vertical, thereby discharging flow stream contents against the inner wall of bottle 300. Tip 328 is preferentially positioned at an angle less than horizontal, and approximately tangential to the inner wall of bottle 310. Adequate direction of tip 328 ensures the majority of mobile phase flow is directed downward, rather than upward on the inner wall of a bottle and that turbulence and shear forces are prevented in the delivery stream. Through tip 328, the mobile phase flow stream follows a descending spiral path. Further separation of remaining supercritical gas from the liquid phase is enhanced as the flow swirls down bottle wall. The swirling effect also delivers liquid sample fractions into bottle 310 very gently. Gasses and vapors, such as those emanating from CO2 and methanol, can exit through discharge line 304 that leads to waste system 306.

[0113] Unattended runs may require monitoring fill levels in bottles 310, so that when liquid reaches a threshold level, the mobile phase flow path is redirected to a different bottle capable of receiving flow. It is obvious that a conventional method for monitoring fill levels is to visually observe the height of liquid in each bottle 310 through access panel 322 as long as the bottle itself is transparent. However, for long sample runs, using laboratory personnel to watch a bottle fill with liquid is inefficient. The sample collection system of the present invention can be automated by controller 332. Controller 332 records data from detector 216, injection valve 212, and distribution valve 22. A controller 322 may calculate an estimate of the amount of liquid phase recovered from the injection through estimation or physical detection based on mobile phase's volumetric flow rate and the volume of each discrete sample injection.

[0114] Controller 322 then tracks elution volumes that are deposited into a collection bottle 310, thereby estimating the volume of liquid collected over time. When the estimated volume for a specific time period is retained, an alarm is triggered and flow is stopped. An alternative process uses a mechanical means, such as sensors in each bottle that detect a physical fluid level and signals controller to redirect flow via the flow valve to an empty collection bottle or to stop flow.

[0115] Discrete sample volumes that are eluted through column 214 appear as concentration peaks on a chromatograph. Controller 332 automates sample injection valve 212 to inject a discrete sample volume into the mobile phase. To accomplish rapid injection, elution, and collection, the system flow variables should remain as constant as possible in a steady operating state, under approximate isocratic conditions. Although steady-state flow conditions in SFC are more difficult to control than those in liquid chromatography, maintaining temperature, pressure, and flow stream composition as consistent as practicable are advantageous to reproduce elutions at predictable time periods in the flow stream.

[0116] System controller 332 saves elution periods of sample solutes through column 214 that are targets of collection processes. The time period between injection of a discrete sample volume through the injection valve 212 and the appearance of the leading edge of the targeted solute's peak concentrations through column 214 and detector 216 is measured and recorded. For example, a sample injection may produce peaks at five minute intervals after injection. If a first peak appears at a five minute time period from injection and the elution lasts for a two minute time period, the separation times would normally allow one sample injection approximately every eight minutes. However, injections are optimized for rapid collection of eluted solutes. In the example, controller 332 injects samples every two minutes for elutions of specific solutes, regardless of whether the entire chromatographic results of the previous injections have completed their runs. This process results in an improvement of three to ten times over the rate at which samples may be separated and retained by the collection system over prior methods. When not collecting targeted solutes, the flow stream is directed to waste collection system 306. An alternative process sends a start/stop signal to signal the distribution valve 22 based strictly on detection of peaks rather than a timetable of peak appearance in the detector. However, the preferred alternative embodiment provides rapid sample injection and collection process through timing of sample injections that elute under consistent operating conditions.

[0117] Because many varying and different embodiments may be made within the scope of the inventive concept herein taught, and because many modifications may be made in the embodiments herein detailed in accordance with the descriptive requirements of the law, it is to be understood that the details herein are to be interpreted as illustrative and not in a limiting sense. 

What is claimed:
 1. A process for collecting samples from a flow stream containing a mixture of highly compressed gas, compressible liquid or supercritical fluid and a relatively incompressible liquid comprising: controlling the pressure, temperature and velocity of said flow stream to enhance separation processes of a monophasic fluid mixture into separate gaseous and liquid phases; expanding said flow stream by directing said flow stream through an expansion space; retaining said liquid phase in a collection device.
 2. The process of claim 1, wherein: said step of expanding said flow stream is provided by directing said flow stream through large bore tubing having internal diameter adequate to slow the linear speed of said flow stream.
 3. The process of claim 1, wherein: said step of expanding said flow stream is provided by directing said flow stream through a chamber having internal space adequate to slow the linear speed of said flow stream.
 4. The process of claim 1, wherein: said step of retaining said liquid phase in a collection device includes using a discrete collection container to receive said liquid phase, wherein said container has an exit port for discharging waste products.
 5. The process of claim 1, wherein: said flow stream discharges from a flow line inside said collection device at an angle less than horizontal and at a tangential angle to the inner wall of said container.
 6. The process of claim 1, further comprising: detecting the volume of said liquid phase in said collection device and stopping said flow stream filling said device when said liquid phase reaches a threshold level.
 7. The process of claim 1, further comprising: performing said process under approximate isocratic conditions.
 8. The process of claim 1, further comprising: injecting samples into said process at a frequency such that a second sample injection begins elution of a sample solute after a first sample injection completes elution of the same said solute within the first injection, but prior to said first sample completing an entire chromatographic process.
 9. The process of claim 8, wherein: determining the frequency of said sample injections comprises detecting said eluted solutes in said flow stream, determining time periods for elution of said solutes from time of injection to the beginning of elution, determining time periods from start to finish of an eluted solute concentration peak, and automatically collecting said liquid phase containing said eluted solutes from said repetitive sample injections into said collection device based on said time periods.
 10. The process according to claim 1, further comprising: retaining liquid phase in said collection device according to a start/stop signal from a detection device that is sent through a system controller for each of said sequential series of injections.
 11. A process for collecting samples from a flow stream containing a mixture of highly compressed gas, compressible liquid or supercritical fluid and a relatively incompressible liquid comprising: injecting samples into said flow stream; controlling the pressure, temperature and velocity of said flow stream to enhance separation processes of a monophasic fluid mixture into separate gaseous and liquid phases; expanding said flow stream by directing said flow stream through an expansion space in a flow line carrying said flow stream; retaining said liquid phase in a collection device.
 12. The process of claim 11, wherein: said step of expanding said flow stream is provided by directing said flow stream through large bore tubing having internal diameter adequate to slow the linear speed of said flow stream.
 13. The process of claim 11, wherein: said step of expanding said flow stream is provided by directing said flow stream through a chamber having internal space adequate to slow the linear speed of said flow stream.
 14. The process of claim 11, wherein: said step of retaining said liquid phase in a collection device includes using a discrete collection container to receive said liquid phase, wherein said container has an exit port for discharging waste products.
 15. The process of claim 11, wherein: said step of injecting samples occurs at a frequency such that a second sample injection begins elution of a sample solute after a first sample injection completes elution of the same said solute within the first injection, but prior to said first sample completing an entire chromatographic process.
 16. The process of claim 15, wherein: determining the frequency of said sample injections comprises detecting said eluted solutes in said flow stream, determining time periods for elution of said solutes from time of injection to the beginning of elution, determining time periods from start to finish of an eluted solute concentration peak, and automatically collecting said liquid phase containing said eluted solutes from said repetitive sample injections into said collection device based on said time periods.
 17. A system for collecting samples from a flow stream containing a mixture of highly compressed gas, compressible liquid or supercritical fluid and a relatively incompressible liquid comprising: a flow line creating a space wherein said flow stream moving through said line is expanded and the linear velocity of said flow stream is slowed; a collection device located downstream of said space in said flow line, wherein liquid phase from said flow stream is retained.
 18. The system of claim 17, wherein: said space in said flow line is provided by a large bore tubing having internal diameter adequate to slow the linear speed of said flow stream.
 19. The process of claim 17, wherein: said space in said flow line is provided by directing said flow stream through a chamber having internal space adequate to slow the linear speed of said flow stream.
 20. The process of claim 17, wherein: said collection device includes a discrete collection container to receive said liquid phase, wherein said container has an exit port for discharging waste products.
 21. A system for collecting samples from a flow stream containing a mixture of highly compressed gas, compressible liquid or supercritical fluid and a relatively incompressible liquid, comprising: an injection valve injecting discrete samples into said flow stream; a separation device to elute solutes of said samples; a detector to detect concentrations of said solutes in said flow stream; a phase separation stage to control the pressure, temperature and velocity of said flow stream to enhance separation processes comprising a series of heaters and transfer lines to separate a monophasic flow stream into liquid and gas phases; an expansion space in the flow stream that is sized to create a point of expansion of said flow stream and wherein the linear velocity of said flow stream is slowed; at least one collection device to retain said liquid phase.
 22. The system of claim 21, wherein: said space in said flow line is provided by a large bore tubing having internal diameter adequate to slow the linear speed of said flow stream.
 23. The process of claim 21, wherein: said space in said flow line is provided by directing said flow stream through a chamber having internal space adequate to slow the linear speed of said flow stream.
 24. The process of claim 21, wherein: said collection device includes a discrete collection container to receive said liquid phase, wherein said container has an exit port for discharging waste products. 